Yeast (YAC) and Human (HAC) Artificial Chromosome Clones

Abstract

Artificial chromosome vector systems in yeast and bacteria have facilitated the increasingly rapid pace of mapping and sequencing of complex genomes. Traditional bacterial cloning systems have remained important for the study of relatively short clones, but for the cloning of very large deoxyribonucleic acid (DNA) segments yeast artificial chromosomes (YACs) have completely replaced earlier bacterial systems, including lambda phage‐based cosmids. YACs comprise cloned DNA fragments ranging from 50 kb to more than 1 million base pairs, along with sequences that render them capable of growth in yeast or bacteria. YACs provide the additional advantage of permitting direct isolation of a targeted genomic region as a circular molecule from complex genomes by transformation‐associated recombination. Moreover, YAC cloning has allowed the propagation of large tandem repeat arrays and entire bacterial genomes. By taking advantage of the efficient yeast recombination system, YAC clones can be further modified and used for functional studies of full‐length genes and for the study of huge centromeric DNA repeat regions that are not yet analysed or included in the genome assemblies of human and other organisms.

Key Concepts:

  • Basic structure of vectors used in the construction of artificial chromosomes that can replicate in yeast, bacteria and human cells.

  • Utilities of artificial chromosomes.

  • Isolation of complete genes and large DNA segments, including repetitive chromosomal regions.

  • Targeted recombination‐based cloning of genes and highly repetitive regions.

  • Reconstruction of large DNA regions by recombination among multiple YAC clones.

Keywords: YACs; BACs; PACs; artificial chromosomes; cloning vectors; HAC‐based vectors

Figure 1.

Generalised features of a typical YAC cloning vector and cloning steps. The YAC vector has yeast selectable auxotrophic markers for tryptophan (TRP1) and uracil (URA3) biosysnthesis. The centromere (CEN4) sequence is derived from yeast centromere 4. Cleavage with BamHI and EcoRI releases the two arms of the YAC vector terminating in T. pyriformistelomere (TEL) sequences associated with one of the selectable markers and disrupts the SUP4 gene that suppresses the ADE4 mutation. The stuffer fragment released by digestion with BamHI between the telomeres is removed during purification of the arms. The genomic DNA is digested with EcoRI in the presence of EcoRI methylase to prevent excessive digestion, fractionated by pulsed field gel electrophoresis (or zonal sedimentation through a sucrose gradient) and ligated to the arms and transformed into S. cerevisiae strain AB1380. Mutation in the ADE4 gene in the host strain yields transformed colonies that are CEN4 in the absence of the SUP4 gene on a selection medium lacking uracil and tryptophan. Such colonies are further purified and the presence of insert verified. © US govt.

Figure 2.

Principles of transformation‐associated recombination (TAR) cloning. A BAC‐based vector containing yeast centromere (CEN) and selectable marker (HIS3) sequences is cleaved with a restriction enzyme at a unique site placed between the ‘hooks’ (DNA sequences of at least 200 bp at the edges of the region to be cloned). The digested vector is transformed into yeast along with high molecular weight genomic DNA. The ‘hooks’ pair with the corresponding sequences in genomic DNA, and recombination produces the TAR clone. The cells are plated and recombinants that grow depend on the presence of replication origins in the cloned DNA, thus ensuring that the DNA between the ‘hooks’ is rescued. Note that this vector system rescues genomic DNA in a circular form; unlike the YAC construction it does not require the presence of telomere sequences, and discourages cocloning events; in fact, no cocloned TAR‐derived YAC has been observed. © US govt.

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Further Reading

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual, 2nd edn, vol. 1, pp. 22–358 . Plainview, NY: Cold Spring Harbor Laboratory Press.

Web Links

For a list of sequenced organisms refer to http://www.ncbi.nlm.nih.gov/genome

For human genome sequence assembly http://www.ncbi.nlm.nih.gov/projects/genome/guide/human/index.shtml

Vector Information. For a description of PAC/BAC vectors, their sequences and library resources. http://www.chori.org/bacpac/vectors.htm

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How to Cite close
Nagaraja, Ramaiah, Kouprina, Natalay, Larionov, Vladimir, and Schlessinger, David(May 2013) Yeast (YAC) and Human (HAC) Artificial Chromosome Clones. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005347.pub2]